Project Summary Anopheles coluzzii is one of most important vectors in sub-Saharan Africa. One reason for this is its high preference for human hosts. Like all organisms, An. coluzzii has an internal clock that controls much of its biology and behavior. For example, An. coluzzii is active in the evening and at night, and mating, host seeking, sugar feeding and oviposition takes places during this time. For example, spontaneous flight activity of this mosquito is all but absent during the day but shows two strong peaks of activity during the night: one in the early evening and one towards the end of the night. Previous work has also shown that the expression of many genes is controlled by the internal clock. This includes genes involved in olfaction, immunity and insecticide resistance. The internal clock consists of several genes, including the genes clock and cycle, that are expressed rhythmically. Together with other genes they form a feedback loop that cycles approximately every 24 hours. In Aedes mosquitoes, which are active at night, shutting down this internal clock by disabling the cycle gene results in much reduced host seeking, longer development times and reduced survival. Because the internal clock affects day-time and night-time mosquitoes differently, the results from Aedes cannot be extrapolated to Anopheles. Therefore, to examine to what extent shutting down the internal clock affects the behavior, development and survival of An. coluzzii, we will use CRISPR-Cas9 to disable the clock or cycle gene. We will evaluate how this affects spontaneous flight activity, host seeking, mating success, as well as developmental rate and survival. This will tell us to what extent this mosquito?s behavior is controlled by the internal clock vs external conditions (e.g light/dark). It may also provide a way to modify mosquito behavior, e.g. by reducing host seeking, to reduce its ability to transmit malaria.